Calcium/calmodulin-dependent protein kinase II controls alpha5beta1 integrin-mediated inside-out signaling.
Fibronectin binding on alpha5beta1 integrin is strictly dependent on intracellular calcium. Using an in vitro assay, we previously found that either calcineurin inhibitors or a blocking calcineurin monoclonal antibody added to cell lysates completely abolished the fibronectin/integrin interaction, which suggested that the activity of calcineurin, a calcium/calmodulin-dependent phosphatase, was required to counteract some kinase activity and maintain the high affinity state of alpha5beta1. In this paper, we show that blocking of the calcium/calmodulin kinase II (CaMKII) activity with the specific inhibitor KN-62 or with its pseudosubtrate Autocamtide-2 preserved the high affinity state of the integrin even under experimental conditions that inhibit calcineurin. Conversely, the addition of purified CaMKII to the cell lysate inhibited alpha5beta1 binding to fibronectin in vitro. Consistent with these results, cell adhesion on fibronectin was stimulated by KN-62. Moreover, Scatchard analysis of fibronectin binding on CHO cells revealed that KN-62 decreased the Kd value from 0.3 to 0.05 microM. Finally the expression of exogenous constitutively active CaMKII resulted in a dramatic defect in cell adhesion with no significant modification in alpha5beta1 cell surface expression. In summary our results demonstrate that CaMKII controls the affinity state of the integrin alpha5beta1 in vitro and in living cells.
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LEDAC/UMR CNRS-UJF 5538, Institut Albert Bonniot, Faculté de Médecine, F38706 La Tronche Cedex, France.
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D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D065095:Calcineurin Inhibitors; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects; D006224:Cricetinae; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005353:Fibronectins / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D017469:Receptors, Fibronectin / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D005353:Fibronectins; D010455:Peptides; D017469:Receptors, Fibronectin; C085903:autocamtide-2; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases